Split-GFP tagging and live imaging of hair cell proteins

毛细胞蛋白的 Split-GFP 标记和实时成像

• 批准号: 10438419
• 负责人: Jung-Bum Shin
• 金额: $ 24.23万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10438419
• 关键词: Address; Adopted; Affect; Behavior; Cell physiology; Cells; Cellular biology; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; DNA; Development; Diffusion; Electroporation; Equilibrium; Fluorescence; Fluorescence Microscopy; Gene Expression; Genes; Goals; Gold; Hair; Hair Cells; Hearing; Image; Investigation; Kinetics; Knock-in; Knock-in Mouse; Labyrinth; Length; Link; Mediating; Methods; Modernization; Molecular; Morphologic artifacts; Motor; Movement; Mus; Pillar Cell; Property; Proteins; Role; Sensory Receptors; System; Techniques; Testing; Transfection; Transgenic Mice; USH1C gene; Virus; fluorescence imaging; gene gun; genome editing; genomic locus; hearing impairment; insight; interest; live cell imaging; nanoscale; overexpression; reconstitution; tool; transmission process

Over the past few decades, the hair cell field has gained great insight into the inner workings of hair cells, the sensory receptors that mediate our sense of hearing and balance. The molecular identity of many factors important for hair cell development and function, and their significance for hearing loss, have been elucidated. Despite this progress, the study of hair cells presents many challenges. In particular live-cell imaging and tracking of proteins, important pillars of cell biology studies, have been especially difficult, mainly due to limited means to transfect and culture hair cells. Considering that the essence of hair cell function is micro- and nanoscale movement, investigations of the dynamic properties of proteins is crucial for a complete understanding of hair cell function. The goal of this proposal therefore is to develop a tool that makes imaging of protein movements in living hair cells more accessible. The gold standard technique for tracking protein movement is live-cell imaging of tagged proteins. Traditionally, this is achieved by expression of exogenous DNA constructs fused to fluorescent proteins, delivered to cells by gene gun, electroporation or viruses. Transgenic mice also have been employed. These approaches, however, are potentially confounded by overexpression artifacts and low transduction/transfection efficiency. Knock-in (KI) of genetically-encoded fluorescent tags into the endogenous gene loci has become more tractable through modern genome editing methods, but KI of large DNA segments, such as the full-length GFP coding sequence, remains difficult and has the potential to affect gene expression. To address these challenges, we plan to develop a mouse tool kit that streamlines fluorescent tagging of proteins for localization and live cell imaging. To this end, we adopted the Split-GFP strategy. In this two- component system, the endogenous gene of interest is genetically tagged with a small part of GFP (“GFP11”). Co-expressing the remaining (non-fluorescent) portion of GFP (“GFP1-10”) reconstitutes GFP fluorescence, allowing imaging by fluorescence microscopy. Due to the small size of the GFP11 fragment (48 bps), CRISPR- mediated KI into the endogenous loci is highly efficient. In preliminary studies, we confirmed that the Split-GFP approach faithfully recapitulates the endogenous localization of the cuticular plate protein LMO7, by delivering AAV-packaged GFP1-10 into the hair cells of Lmo7-GFP11 KI mice. To circumvent AAV-mediated delivery, we propose to generate a transgenic mouse line that expresses the GFP1-10 fragment. Founders have already been obtained and germline transmission was confirmed (SA1). This system will be tested on live-imaging tasks of increasing difficulty: In SA2, we will study the diffusion kinetics and movement of LMO7 in the hair cell. In SA3, challenging the sensitivity limits of this system, we will investigate the dynamics of the tip link motor complex component USH1C in the hair bundle.

在过去的几十年里，毛细胞领域对毛细胞的内部工作原理有了很大的了解